The development of new forms of therapeutics which use macromolecules such as proteins or nucleic acids as therapeutic agents has created a need to develop new and effective means of delivering such macromolecules to their appropriate cellular targets. Therapeutics based on either the use of specific polypeptide growth factors or specific genes to replace or supplement absent or defective genes are examples of therapeutics which may require such new delivery systems. Clinical application of such therapies depends not only on efficacy of new delivery systems but also on their safety and on the ease with which the technologies underlying these systems can be adapted for large scale pharmaceutical production, storage, and distribution of the therapeutic formulations.
Gene therapy has become an increasingly important mode of treating various diseases. The potential for providing effective treatments, and even cures, has stimulated an intense effort to apply this technology to diseases for which there have been no effective treatments. Recent progress in this area has indicated that gene therapy may have a significant impact not only on the treatment of single gene disorders, but also on other more complex diseases such as cancer.
Success of a gene therapy protocol largely depends upon the vehicle used to deliver the gene. A variety of means exist to introduce a gene inside the cell including physical means such as microinjection (Capecchi, M. R. Cell (1980) 22:479-485), electroporation (Pacqereau, L. et al. Anal. Biochem. (1992) 204:147-151) and particle bombardment (Yang, N. -S. et al. Proc. Natl. Acad. Sci. USA (1990) 87:9568-9572)), biological means such as viruses (Ferry, N. et al. Proc. Natl. Acad. Sci. (1991) 88:8377-8381) and chemical means such as calcium phosphate (Wiegler, M. et al. Cell (1977) 11:223-232), DEAE dextran (Ishikawa, Y. et al. Nucl. Acid Res. (1992) 20:4367-4370), polylysine (Wu, G. Y. et al. J. Biol. Chem. (1988) 263:4429-4432) and cationic liposomes (Felgner, P. L. et al. Proc. Natl. Acad. Sci. (1987) 84:7413-7417)). Clinical application of such therapies depends not only on the efficacy of new delivery systems but also on their safety and on the ease with which the technologies underlying these systems can be adapted for large scale pharmaceutical production, storage, and distribution of the therapeutic formulations. Thus, an ideal vehicle for the delivery of exogenous genes into cells and tissues should be highly efficient in nucleic acid delivery, safe to use, easy to produce in large quantity and have sufficient stability to be practicable as a pharmaceutical.
Non-viral vehicles, which are represented mainly by cationic lipids, are one type of vehicle which have, for the following reasons, been considered for use in gene therapy. First, the plasmid DNA required for liposome-mediated gene therapy can be widely and routinely prepared on a large scale and is simpler and carries less risk than the use of viral vectors such as retroviruses. Second, cationic lipids are less toxic and less immunogenic than viral vectors and the DNA complexed with the lipids is better protected from degradation by nucleases. Third, liposome-mediated gene delivery, unlike retroviral-mediated gene delivery, can deliver either RNA or DNA. Thus, DNA, RNA or an oligonucleotide can be introduced directly into cells using cationic liposomes.
Among the numerous cationic amphiphiles which have been referred to as useful for transfecting nucleic acids into cells are cationic derivatives of cholesterol. For example, cholesterol (4'-trimethylammonio) butanoate (ChOTB) contains a trimethylammonium group connected to the 3'-hydroxyl group of cholesterol via a butanoyl spacer arm and cholesterol hemisuccinate choline ester (ChOSC) contains a choline moiety connected to the 3'-hydroxyl group via a succinyl spacer arm. However, the transfection activities of these amphiphiles are generally weak. (Leventis, R. et al. (1989) Biochim. Biophys. Acta., 1023:124-132)
Epand et al. (U.S. Pat. No. 5,283,185) describe cationic derivatives of cholesterol in which primary, secondary, tertiary or quaternary alkyl ammonium groups are linked to cholesterol via the 3-hydroxy group. These cationic cholesterol derivatives, including 3.beta.[N-(N', N'-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol), are disclosed to be useful in transfecting nucleic acids into cells.